There has been a recent focus on the role of testosterone in metabolism as well as cardiovascular risk in both men and women. Studies suggest that the relationship of testosterone to metabolic and vascular risk differs in men versus women and are in fact in opposite directions. In men, testosterone deficiency has been associated with higher rates of the metabolic syndrome, insulin resistance, type 2 diabetes, and cardiovascular disease. In contrast, women with a known disorder of androgen excess, polycystic ovary syndrome (PCOS) are at increased risk of metabolic dysfunction including metabolic syndrome and type 2 diabetes. Women with PCOS also have increased risk of intermediate markers of cardiovascular disease but studies with clinical endpoints such as myocardial infarction, congestive heart failure, or stroke are lacking. The consequences of higher testosterone concentrations on women's health in the general population are less clear but are of increasing concern to both patients and clinicians. Additionally, there is concern that lower testosterone levels may be associated with low bone density, poor physical function, sarcopenia, and frailty in women as well as in men. However, understanding the role of testosterone in women's health across the lifespan has been hampered by the inability to measure testosterone accurately and reliably in the relatively low concentrations in women compared to men. A recent position statement by the Endocrine Society's Expert Panel acknowledged that the common currently used radio-immunoassays that were developed to measure testosterone in men are not reliable for measuring testosterone in women. The overall objective of this proposal is to characterize circulating testosterone in women across a broad age range from a community-based population using state of the art assay methodology for measuring testosterone in women, liquid chromatography tandem mass spectrometry (LC-MS/MS). The Framingham Heart Study (FHS) cohorts are ideally suited for this project given the study's community-based sample population, stored serum, and extensive clinical phenotyping. LC- MS/MS provides accurate and precise measurement of testosterone concentrations in the low range found in women as well as circulating estradiol concentrations. We will assess the association between circulating testosterone levels and health outcomes in ~4500 women, focusing primarily on metabolic and cardiovascular disease but also physical function and bone density, in cross-sectional and longitudinal analyses over a 5-10 year period. The influence of testosterone will be evaluated in the context of circulating estradiol levels given the long-standing concern for estrogen effects on vascular and bone disease in particular. The interaction between testosterone and metabolic function is increasingly recognized as an important and potentially modifiable CVD risk factor. The clinical consequences of low testosterone in women are not well characterized at this time. Greater knowledge about the health consequences of both low and high testosterone in women, a potentially modifiable hormonal factor, may facilitate the identification of high risk individuals as well as the development of effective risk reduction strategies for women across a broad age range. PUBLIC HEALTH RELEVANCE: The clinical impact of estrogen levels on women has been a significant focus of medical research for a long time, particularly in regard to cardiovascular disease, osteoporosis, and cancer. However, little is known about the effect of testosterone on women's health in the general population but the effects of androgens are of increasing concern clinically. Women with polycystic ovary syndrome, a known disorder of androgen excess, are at increased risk for metabolic syndrome, type 2 diabetes, dyslipidemia and hypertension, raising concern for increased cardiovascular disease risk as well. There is also increasing evidence of adverse health conditions in women with low androgen states including low bone density, depression and potentially poor physical function, sarcopenia, and frailty. Thus, there may be a critical range of testosterone levels in women associated with health. Determination of the adverse health effects of androgen excess and deficiency is crucially dependent upon knowledge of normal androgen states in women across the lifespan. Our overall objective is to characterize circulating testosterones in women across a broad age range from a community- based population;the Framingham Heart Study (FHS) cohorts are ideally suited for this project given the study's community-based sample population, stored serum, and extensive clinical phenotyping. Testosterone will be measured using state of the art assay methodology, liquid chromatography tandem mass spectrometry (LC-MS/MS) that provides accurate and precise measurement of testosterone concentrations in the low range found in women. We will assess the association between circulating testosterone levels and health outcomes in women, focusing on metabolic and cardiovascular disease but also physical function and bone density.